Let a good idea filter through

With unremitting pressure on analytical performance, lab efficiency and cost management, it is easy to forget that the basics of sample prep can make a huge difference to your liquid chromatography. Here, Xiaomi Xu discusses matrix effects, and the impact of particulates and impurities 

The statistics are telling. Sample processing accounts for around one-third of errors in chromatographic analysis. And pre-separation work typically takes up more than 60% of time spent on sample analysis. Precise, efficient sample preparation is therefore an essential component for success – ensuring sample integrity and removing matrix interferences. These matrix interferences may otherwise have deleterious effects on analyte separation and detection, as well as minimising maintenance and troubleshooting of the chromatography hardware.

Routines must be as straightforward as possible, cost effective, and be compatible with analytical goals and a laboratory’s established workflows for LC, HPLC, and UHPLC

Where complex samples are to be analysed – for example those samples with a lot of potential matrix interferences or analytes present at trace levels – accurate, productive chromatography demands careful sample preparation. Routines must be as straightforward as possible, cost effective, and be compatible with analytical goals and a laboratory’s established workflows for LC, HPLC, and UHPLC. They must be easy to use, or amenable to automation – after sample processing, operator error is the next largest reason for suboptimal chromatography.

A list of the consequences of poor sample prep is a long one, including; plugged columns, increased backpressure, retention time shift, resolution loss, poor reproducibility between runs, false positives and negatives in trace-level analysis, and shorter column lifetime. These consequences can be divided into two broad categories – physical effects and chemical interferences. Many labs will simply rely on a simple ‘dilute and shoot’ approach. The assumption is that any particulates and matrix interferences are diluted out to a level where they do not impact chromatography performance. For some samples, it is an acceptable strategy, but in other cases, sample prep can make a significant impact. Table 1 shows the most commonly used sample preparation methods.

[box type="shadow" ]Table 1: The most commonly used sample preparation methods

• Filtration: This technique is ideal for a multitude of organic or aqueous samples containing particulates, which could cause instrument blockage or downtime. Protein precipitation-based filtration techniques are also available, and offer a fast and generic way of removing proteins from a range of samples. Some versions of this technique also include a lipid depletion property, which also helps remove common lipids.
• Solid supported liquid extraction (SLE): Ideal for aqueous samples containing nonpolar analytes, this technique offers advantages over standard liquid-liquid extraction methods. These include support for automation, lower solvent usage, and better recoveries and precision by removing issues with emulsions that are often formed when performing liquid-liquid extraction.
• Solid phase extraction (SPE): When high sample cleanliness is required, this most selective sample preparation technique ensures that analysis is accurate. SPE offers the best combination of interference removal and analyte enrichment.
• Dispersive SPE, including methods like QuEChERS: A streamlined sample prep approach that has been widely adopted for multiclass, multi-residue analysis in complex matrices, including food and environmental samples. The name stands for "Quick, Easy, Cheap, Effective, Rugged, and Safe."

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By removing impurities and particulates, sample filtration helps to prevent column failure, and is arguably a vital front line approach to protecting your column, chromatography, and ensuring column longevity

Let’s look more closely at simple physical interferences. Particulates can create blockages in the column and cause mechanical issues within instruments – this is not always noticeable and can affect chromatography and reproducibility. First, costly instrument downtime, sample reruns and an increased need for system cleaning are the most obvious result. In addition, analyte detection can be suppressed, baseline noise increased, and sensitivity reduced. Results can become unreliable and difficult to interpret, with poor peak shapes, interferences and co-elutions leading to the need to repeat analysis. By removing impurities and particulates, sample filtration helps to prevent column failure, and is arguably a vital front line approach to protecting your column, chromatography, and ensuring column longevity. Filtration products are available in a wide variety of membrane types and pore sizes to suit various samples and analysis needs.

So, how do you go about choosing the right syringe filter for your application? For most applications, there are three simple questions to ask:

• What type of sample (analytes and matrices) needs analysing?
• What is the volume of my sample?
• What is the particle size of the column I am using?

Using online product guides can help to make sure that you choose the right syringe filter for your analytical needs. Low protein binding and cleanliness are important features of a syringe filter’s performance in this application. Polyethersulfone (PES) and polyvinylidene fluoride (PVDF) membranes are typically used for biological sample filtration and can provide low protein binding. PVDF membranes are widely used for filtering biological samples targeted to protein and peptide analysis. However, PVDF syringe filters can cause contamination during filtration, which can damage the integrity of samples. Common protein analysis techniques may not observe these contaminants due to limited detector sensitivity and selectivity. Therefore, it is critical to use the cleanest filter to prevent the introduction of any contamination during sample preparation. In one study¹, Agilent’s Captiva Premium PES syringe filters were evaluated and compared with other PES and PVDF syringe filters for filtration recovery, targeted protein analysis, and chemical cleanliness. Captiva premium filters provided superior recoveries (>97%) over other PDVF syringe filters for proteins and peptides, including monomers and dimers. The results demonstrate how Captiva premium filters can firmly support the analysis of low-level protein samples with minimal loss during filtration.

Methods for the analysis of multiple residues of pesticides in food have received a great deal of attention, particularly as scares regarding contaminants in our food supply have grown

Beyond filtration, many labs use techniques such as Solid Phase Extraction (SPE) or Dispersive SPE (dSPE) methods to remove unwanted matrix components that may compromise trace-level detection. Sample preparation and analysis of trace compounds in complex food matrices has always been considered a major challenge. Methods for the analysis of multiple residues of pesticides in food have received a great deal of attention, particularly as scares regarding contaminants in our food supply have grown. Traditional sample preparation in this application used liquid-solid extraction, liquid-liquid extraction and more recently, solid-phase extraction (SPE) to extract unwanted matrix components ahead of identifying and quantifying pesticides of interest.

Around 10 years ago, a new technique was developed, which built on the familiar extraction procedures. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) was developed as a simple yet effective method for isolating trace pesticides from many fruits and vegetables. With its simple techniques and effective matrix removal, QuEChERS has become the de facto standard for food safety analysis and has also been adopted for the sample prep for a wide variety of other contaminants. The method involves two steps. An extraction step (based on partitioning via salting-out extraction involving equilibrium between an aqueous and an organic layer) and a dispersive solid-phase extraction (dSPE) step. The later involves further clean up using various combinations of salts and sorbents to remove interfering matrices.

Today, QuEChERS methods for hundreds of pesticides in many fruits, vegetables, meat, and dry materials such as beans and nuts have been published. Official methods are now available and a standardisation of the technique on a worldwide basis is taking place. In the United States, the Association of Official Analytical Chemists (AOAC) has published its 2007.01 Method while the European equivalent, the EN 15662 2007, uses similar methodology. Importantly, routine labs can now access a comprehensive range of prepared extraction and dispersive kits that enable straightforward implementation of the standard methods. Simply choose the extraction kit based on the method being used (AOAC or EN). Then, add the dispersive kit depending on the type of sample being processed. In the same way as noted for filtration, online product guides make these choices easy to make.

However, if lipids are not removed properly they can collect in the MS source, increasing the need for instrument maintenance and resulting in unnecessary downtime

Lipids are a challenging matrix to remove. If not effectively removed, the lipids build up in the instrument and column, contributing to downtime and reducing column lifetime. Lipids can also suppress ions, which reduces analyte sensitivity and reproducibility in mass spectrometry applications. Due to its high level of sensitivity and specificity, GC/MS and LC/MS are often used for food analysis. However, if lipids are not removed properly they can collect in the MS source, increasing the need for instrument maintenance and resulting in unnecessary downtime. Against this background, the newly developed sorbent: Enhanced Matrix Removal – Lipid (EMR-Lipid) from Agilent means that high-fat samples such as meat, milk, and fish can now be effectively cleaned up without target analyte loss.

EMR-Lipid provides fast, robust, and effective sample preparation for the analysis of high-fat samples, and is offered in a dSPE kit format. Compared to other matrix cleanup products, EMR-Lipid can be universally applied to the analysis of polar, midpolar, and nonpolar target analytes. EMR-Lipid provides more effective matrix removal from challenging samples and minimizes the need for separate workup and methods. Removing lipids enhances detection sensitivity for trace analysis and as an added benefit, efficient matrix removal using EMR-Lipid dSPE improves system reproducibility and offers longer column lifetime with reduced instrument maintenance².

From mechanical filtration removing particulates, to advanced lipid removal technology for most challenging high-fat samples, there are a wide range of sample preparation techniques to eliminate interferences and matrix effects. These techniques result in cleaner samples for better analysis. Much progress has been made in the quest to reduce errors in chromatographic analysis caused by inadequate sample preparation. But remembering to get the basics right still resonates, and serves to remind us all of the crucial importance of sample preparation to robust, reliable analysis, and overall lab productivity.

Author: Xiaomi Xu, Sample Prep Product Manager, Agilent Technologies

References 1. Zhao L, Duong P. Choosing the best syringe filters for biological sample filtration. Agilent Technologies, Application Note. Publication number 5991-1308EN (2012). 2. Zhao L. Benefits of EMR—Lipid Cleanup with Enhanced Post Treatment on Pesticides Analysis by GC/MS/MS. Agilent Technologies, Application Note. Publication number 5991-6707EN (2016).